Extracellular Vesicles Generated Using Bioreactors and their Therapeutic Effect on the Acute Kidney Injury Model

Abstract

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.

Mesenchymal stem cell culture within perfusion bioreactors incorporating 3D-printed scaffolds enables improved extracellular vesicle yield with preserved bioactivity

“…Coupled with our data, this could mean that the increased EV production we observed in this bioreactor system is a result of fluid dynamics rather than a specific mechanoresponse of the cells. However, in a recent study, while culturing MSCs in a flat-plate perfusion bioreactor, Kang et al found that flow increased intracellular calcium as well as calcium-related proteins, and thus could be a mechanism behind the increase in EV biogenesis [14]. Separately, Guo and colleagues found that the increased MSC EV production present within their perfusion bioreactor was dependent on the expression of yes-associated protein (YAP), a well-known cellular mechanotransducer [12, 56, 57].…”

“…Of the various therapeutic applications of MSCs and MSC EVs elucidated thus far, the role they play in tissue reparative processes (e.g., angiogenesis) has been well-documented [58][59][60][61]. Moreover, it is known that flow-derived shear stress can be used to influence the regenerative properties of both MSCs and their secreted EVs [14]. Across two orthogonal angiogenic assays (i.e., cell migration and tube formation), we found that the angiogenic properties of MSC EVs derived from the perfusion bioreactor were at least as effective as those from flask-grown MSCs (Figure 4B, C).…”

“…However, in addition to flow-derived shear stress, these systems introduce other cues as the topographical, chemical, and mechanical attributes of the membrane itself can affect cellular behavior, which ultimately convolutes the true effectors at play [13]. Recent studies have utilized simpler systems (e.g., flat-plate bioreactors) [14], allowing a more precise approach to understanding and harnessing flow-derived shear stress but still lack the tunability necessary for dynamic manufacturing and exploratory research needs.…”

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

“…Specifically, bioreactorbased culture allows for the implementation of physiological parameters, including flow-derived shear stress, and has been shown to significantly increase EV production across numerous studies [5][6][7][8][9][10][11][12][13][14]. Importantly, the shift from static to dynamic culture is also known to cause dramatic changes in the EV therapeutic profile [9,12,[14][15][16]; in some cases elucidating EV effects that were not present when utilizing traditional flask culture [15]. This phenomenon of altered EV bioactivity can also be observed when implementing different EV isolation strategies such as tangential flow filtration (TFF), ultracentrifugation (UC), or size-exclusion chromatography (SEC) [16,17].…”

“…In terms of EV production, there has been a collective movement to implement dynamic and/or 3D cell culture microenvironments, as these techniques are often more scalable than traditional flask culture and can produce EVs that resemble those within the physiological niche [4]. Specifically, bioreactorbased culture allows for the implementation of physiological parameters, including flow-derived shear stress, and has been shown to significantly increase EV production across numerous studies [5][6][7][8][9][10][11][12][13][14]. Importantly, the shift from static to dynamic culture is also known to cause dramatic changes in the EV therapeutic profile [9,12,[14][15][16]; in some cases elucidating EV effects that were not present when utilizing traditional flask culture [15].…”